Ligation - (Sep/07/2012 )

Hello everyone
I need suggestion and advice for the ligation
I want to ligate 1,4 kb insert with the vector pPICZaplha A(3,6kb) and transform into E.coli competent TOP10: agter the gel extraction the concentration of my PCR product (insert) is 9,15ng/µl and for the vector is 30,5ng/µl. Are my concentration fine to carry out the ligation?
I am here to take the advice because last time I had more or less the same concentration my both insert and vector but after the transformation I didnt get any colobnies..So can anyone suggest me the correct manner to carry out the proper ligation.. i am stuck up with the 4 months.. Kindly suggest me how should I carry it out to get the result.
Last time i had the molar ration of 2:1,3:1,4:1 but noone of them worked..
Waiting for the quick reply thanks and regards

-siddharthsameer-

It is a bit too low for my taste but it should work:
take 8 uL of your insert; 0.5 uL of your backbone; 1 uL of 10X T4 DNA ligase buffer, 0.5 T4 DNA ligase (if NEB otherwise tell me the company you have and I check their recommendations and come back to you). I incubate this at room temperature for 1 hour. Then transform 4 uL of this in my competent cells if chemical competent cells and 2 uL if electrocompetent and plate 100 uL of the transformant and 900 uL of the transformant (spin down, throw away most of the liquid except for ~100 uL that you use for resuspending the cell pellet) on 2 agar plates and grow them ON at 37oC (minimum of 16 h). Colony PCR tomorrow.

Would you mind sharing with me your transformation method for me to check some things?

Andreea

-ascacioc-

Could you tell us how you have prepared your plasmid and insert? Usually "ligation" problems are really problems with the insert and vector preparation, or with transformation.

-phage434-

ascacioc on Fri Sep 7 11:55:45 2012 said:

It is a bit too low for my taste but it should work:
take 8 uL of your insert; 0.5 uL of your backbone; 1 uL of 10X T4 DNA ligase buffer, 0.5 T4 DNA ligase (if NEB otherwise tell me the company you have and I check their recommendations and come back to you). I incubate this at room temperature for 1 hour. Then transform 4 uL of this in my competent cells if chemical competent cells and 2 uL if electrocompetent and plate 100 uL of the transformant and 900 uL of the transformant (spin down, throw away most of the liquid except for ~100 uL that you use for resuspending the cell pellet) on 2 agar plates and grow them ON at 37oC (minimum of 16 h). Colony PCR tomorrow.

Would you mind sharing with me your transformation method for me to check some things?

Andreea

Hello andreaa
sorry for the late reply as my internet is kaputt .. I am using T4 DNA ligase from NEB, I dont mind sharing my transformation protocol but i can only send it to you once I go to the lab tomorrow, i will attach and send it to you... You know my supervisor is asking me to make 40microlitre total volume for the ligation as the last time.and asking for the incubation overnight,, i am very skeptical about that...But i do the transformation through electroporation...
sorry for being late in r

-siddharthsameer-

phage434 on Fri Sep 7 12:51:45 2012 said:

Could you tell us how you have prepared your plasmid and insert? Usually "ligation" problems are really problems with the insert and vector preparation, or with transformation.

Hello
I prepared my vector through midi prep and the inser was order from the company.... my vector is 3.6kb and insert is 1.4kb..

-siddharthsameer-

Do not worry You are so panicky Relax, we will make this thing work... or at least try hard.

With what your supervisor says: it is not very wrong what she says; I mean it works like her as well, but it is better to do it in small volumes (but take care to have everything mixed nicely on the bottom of the tube not all the components sitting separately on the walls of the tube ) You know what you can do with your supervisor: do it like you in one tube and like her in parallel also and see what works. Only like this you can prove her when she is not right. Also it is a good practice for you as a master student to compare different protocols and choose your own later in life. With cloning is like cooking...a bit of that and a bit of the other thing, while respecting certain rules. This is why people have so many different ways of doing things.

With electroporation: I am not happy with electroporating ligation reactions. It works, but ligation reactions are highly ionic meaning they produce sparks. Take care when you electroporate to use the correct voltage settings for your cuvette gap (check this and tell me what you have). Many people do it wrong here because they use 1.8 kV for all the cuvettes. Moreover, another common mistake is too electroporate too much ligation and get the spark or not a good time constant. You need >5 time constant, check it always after electroporation. It works with 4 as well, but not very well. If it is lower than 4, it comes down to praying for a miracle. Another thing: resuspend immediately in warm SOC media. SOC media contains glucose which inhibits the cells from expressing your protein which makes their life difficult. SOC media increases the transformation efficiency several times over LB media. These are all the important tricks I can think off at the moment, we see tomorrow what else I come up with when I see your protocol.

Andreea

-ascacioc-

You said where your DNA comes from, but not how it is being cut and purified. These are the critical steps, and are often where the problem lies.

Except that I think his KpnI is dead or smth is wrong with it, the rest looked fine But it does not hurt to have a second and third opinion. Especially when it is your opinion.

Andreea

-ascacioc-

ascacioc on Sun Sep 9 10:36:56 2012 said:

Do not worry You are so panicky Relax, we will make this thing work... or at least try hard.

With what your supervisor says: it is not very wrong what she says; I mean it works like her as well, but it is better to do it in small volumes (but take care to have everything mixed nicely on the bottom of the tube not all the components sitting separately on the walls of the tube ) You know what you can do with your supervisor: do it like you in one tube and like her in parallel also and see what works. Only like this you can prove her when she is not right. Also it is a good practice for you as a master student to compare different protocols and choose your own later in life. With cloning is like cooking...a bit of that and a bit of the other thing, while respecting certain rules. This is why people have so many different ways of doing things.

With electroporation: I am not happy with electroporating ligation reactions. It works, but ligation reactions are highly ionic meaning they produce sparks. Take care when you electroporate to use the correct voltage settings for your cuvette gap (check this and tell me what you have). Many people do it wrong here because they use 1.8 kV for all the cuvettes. Moreover, another common mistake is too electroporate too much ligation and get the spark or not a good time constant. You need >5 time constant, check it always after electroporation. It works with 4 as well, but not very well. If it is lower than 4, it comes down to praying for a miracle. Another thing: resuspend immediately in warm SOC media. SOC media contains glucose which inhibits the cells from expressing your protein which makes their life difficult. SOC media increases the transformation efficiency several times over LB media. These are all the important tricks I can think off at the moment, we see tomorrow what else I come up with when I see your protocol.

Andreea

hello andreaa
this is my transformation protocol into competent cells
1, ligation mixture, competent cell on ice provided with 10ml lb media
40µl of competent cell and 1-2µl ligation mixture mixed together and put on ice for 1 minute
micropiulser on the program with 0,2cm cuvette, 40µl cell suspension 2,5 kv, 5 ms
transfer solution into the uvett
add 1ml lb media andtransfer the solution into eppi on place on ice
subsequent incubation at 37 for 60 min
plating the solution 20µl 100µl and 200µl..
this is what i follwed last time...
i also made the competent cell last month that i will be using for the transformation,, and it is electrocompetent cell as far as I think..

-siddharthsameer-

I would definitely test the competence of your cells. Dilute a known plasmid (pUC19 is typical) to the 100 pg/ul level, and transform with your cells and protocol, plating out on amp plates. You should get 10^9 cfu/ug of DNA (if you use 1 ul, then you should get 10^5 CFU transformed). If you plate 20 ul of a 1 ml outgrowth, then you should see 2000 colonies.